Abstract The high-throughput DNA sequencing revolution has brought a match between the scale of nucleic acid quantification and the complexity of human biology. However, despite expectation that similarly-widespread and scalable methods for probing protein function would emerge, most protein functional assays and mutational analyses remain largely low to medium throughput. While multiplexed protein methods exist, they tend to be employed only by a small number of ?specialists? because 1) they have not reached the immense scalability of DNA sequencing methods, and 2) they are technically challenging to implement. Protillion is commercializing a platform for high-throughput protein analysis built directly on the ubiquitous Illumina sequencer, demonstrating a path to bringing protein functional characterization into the high-throughput era. This protein mapping platform (Prot-MaP) enables the generation of tens of millions of clusters of immobilized proteins directly on DNA sequencing flow cell through efficient tethered in-situ transcription and translation. Fluorescence-based protein functional assays can then be performed directly on this protein array, with results quantified by highly sensitive fluorescence imaging. Prot-MaP enables the generation of immense mutational datasets for both peptides and full-length functional proteins, allowing high-throughput analysis of mutational effects based on direct biophysical observations of protein function. Unlike directed evolution methods this platform allows for direct, quantitative measurements of the function of entire protein libraries, and a shorter time-to-result in a single experiment on automated, widely distributed instrumentation. We will deploy the Prot- MaP platform in two different modes ? one using small, ~15 amino acid peptides as affinity reagents, and another displaying larger, ~115 amino acid nanobodies, to characterize the biophysical interactions between T Cell immunoreceptor with Ig and ITIM domains, or TIGIT protein. TIGIT is an ideal example target, as it is highly soluble, commercially available, and has diagnostic (for detecting HIV progression) and therapeutic (in combination with PD1 blockade in cancer immunotherapy) application potential. We plan to analyze our high- throughput data sets 1) to understand the benefits afforded pre-selection of peptides compared to rational engineering of peptide libraries, 2) to identify clades of peptides and nanobodies that form different families of binders, 3) to test the identification of these distinct families that bind distinct epitopes on TIGIT experimentally, and 4) assess the salt and temperature dependence of binding, 5) examine the dependence of specific key peptides to binding using structural information. The identification of nanobodies and peptides that bind distinct regions of TIGIT will open the door to creation of both sandwich-style affinity assays, as well as high affinity ligands (via avidity) for potential therapeutic application. Overall, this work will create and demonstrate a platform for large-scale biochemical characterization and analysis of protein-protein interactions.